CFTR High Expresser BEST4 ⁺ cells are pH-sensing neuropod cells: new implications for intestinal physiology and cystic fibrosis disease

Single-cell RNA sequencing (scRNA-Seq) studies identified a novel subpopulation of epithelial cells along the rostrocaudal axis of human intestine, specifically marked by bestrophin 4 (BEST4), that are enriched for genes regulating pH, GPCR acid-sensing receptors, satiety, cGMP signaling, [Formula: see text] secretion, ion transport, neuropeptides, and paracrine hormones. Interestingly, BEST4⁺ cells in the proximal small intestine express CFTR but have not been widely linked to the previously described CFTR high-expresser cell (CHE) subpopulation in rat and human intestine. ScRNA-Seq studies in rat jejunum identified CHEs and a gene expression profile consistent with human small intestinal BEST4+ and neuropod cells. Protein immunolocalization confirmed that CHEs express CFTR, BEST4, neuropod proteins, high levels of intracellular uroguanylin (UGN), guanylyl cyclase-C (GC-C), and the proton channel otopetrin 2 (OTOP2), and display long basal processes connecting to neurons, confirming that Best4⁺ cells in the proximal small intestine are CHEs. OTOP2, GC-C, and CFTR traffic robustly into the apical domain of CHEs in response to acidic luminal conditions, indicating their roles in luminal pH regulation. In the ΔF508 cystic fibrosis (CF) rat jejunum, the loss of apical CFTR did not affect BEST4 protein expression in CHEs. However, there was an increased abundance of CHE cells in the ΔF508 rat jejunum compared with wild-type animals. Furthermore, ΔF508 rat CHEs expressed higher levels of GC-C at the apical domain compared with wild-type. These data implicate CHEs in intestinal CF disease pathogenesis.<b>NEW & NOTEWORTHY</b> This is the first study to identify CFTR high-expresser cells in the rat small intestine as neuropod cells capable of sensing and responding to luminal pH, and confirms that Best4⁺ cells are CHEs in the proximal small intestine. This study also provides the first characterization of CFTR and relevant mRNA and proteins in CHEs in CF rat models that provide insights into the significance of CHEs to CF intestinal disease.